Enantiomeric resolution of biomarkers in space analysis: Chemical derivatization and signal processing for gas chromatography–mass spectrometry analysis of chiral amino acids

Abstract

The work compares two GC–MS methods for enantioselective separation of amino acids as suitable candidate for stereochemical analysis of chiral amino acids on board spacecrafts in space exploration missions of solar system body environments. Different derivatization reagents are used: a mixture of alkyl chloroformate–alcohol–pyridine to obtain the alkyl alkoxy carbonyl esters and a mixture of perfluorinated alcohols and anhydrides to form perfluoroacyl perfluoroalkyl esters. 20 proteinogenic amino acids were derivatized with the two procedures and submitted to GC–MS analysis on a Chirasil- l-Val stationary phase. The results were then compared in terms of the enantiomeric separation achieved and intensity of MS response. The combination of methyl chloroformate (MCF) and heptafluoro-1-butanol (HFB) allows separation of 14 enantiomeric pairs, five of which display a resolution ( R s ≥ 1.2) supposed to be sufficient to quantify the enantiomeric excess. Three mixtures of trifluoroacetic (TFAA) and heptafluorobutyric (HFBA) anhydrides were combined with the corresponding perfluorinated alcohols – TFE (2,2,2-trifluoro-1-ethanol) and HFB (2,2,3,3,4,4,4-heptafluoro-1-butanol) – to give three different reagents (TFAA–TFE, TFAA–HFB, HFBA–HFB): the derivatives obtained show separation of the same number of proteinogenic amino acids (14 of 20) at a temperature lower than column bleeding limit (200 °C) and 8 of them give a separation with R s ≥ 1.2. Linearity study and limit of detection ( X LOD) computation show that both methods are suitable for quantitative determination of several amino acid diastereomers at trace level ( X LOD ≈ 0.5 nmol as derivatized quantity). Both the procedures were coupled with automatic data handling to increase their suitability for space analysis: the simplified data treatment is especially helpful to handle the low quality data recovered from space experiments and labor and time are saved, as imposed by the space experiments requiring a rapid delivery of the results. To achieve this aim, a chemometric approach based on the computation of the Autocovariance Function (ACVF) was applied to extract information on the enantiomeric pairs present in the sample and the enantioseparation achieved on the chiral column.